Expression and Function of PBXIP1/HPIP in Breast Cancer

1. Supplementary Figure 1 Supplementary Figure 1. A) PBXIP1/HPIP expression in The Cancer Genome Atlas (TCGA) breast dataset. 1. Breast normal (n=61), 2. Invasive breast carcinoma (n=76). P= 1.27E-4. B) PBXIP1/HPIP expression in Turashvili breast dataset. 0. Normal (n=20), 1. Invasive ductal breast carcinoma (n=5). P= 1.0E-4. (Ref: BMC Cancer 2007/03/27). C) PBXIP1/HPIP expression in Zhao breast. 0. Normal (n=3) 1. Invasive ductal breast carcinoma (n=39). (Ref: Mol Biol Cell 2004/06/01). P= 1.0E-4. n= number of samples.

2. Supplementary Figure 2 Supplementary Figure 2.Effect of either HPIP knockdown (by HPIP sh1 or HPIP sh2) on migration of confluent monolayers of MDA-MB231 cells or forced over expression of T7-HPIP on migration of confluent monolayers of MDA-MB231 cells in which HPIP expression had been knocked down by HPIP sh1.Representative phase-contrast images of the site at different time points as indicated after scratch wounding were presented .

3. Supplementary Figure 3 Supplementary Figure 3 . RGD domain is not involved in HPIP-mediated FAK activation. (A) Alignment of RGD motif of HPIP (amino acids 421 to 423) with consensus RGD motifs of known ECM proteins. (B) Effect of RGD mutations in HPIP on FAK activation. Western blot analysis of total lysates from control vector, wild type HPIP (wt), mt1 (HPIP-RAD) or mt2 (HPIP-RGE) transfected in MCF7 cells. (C) Alignment of RGD domain of HPIP protein from different species (top). Western Blot analysis showing the effect of either hHPIP or mHPIP on FAK activation in MCF7 cells. Membranes were probed with indicated antibodies (bottom). H, human; m, mouse.

4. Supplementary Figure 4 Supplementary Figure 4 . Immunofluorescence analysis showing localization of various deletion mutants of HPIP (T7 tagged) (green) and FAK (red) in MDA-MB231 cells. DAPI (blue) stains nucleus.

5. Supplementary Figure 5 Supplementary Figure 5. Time-lapse sequences in MCF7 cells from live cell fluorescence imaging of DsRed-zyxin were first subjected to ImageJ and fluorescence intensities of individual adhesions from background-subtracted images were measured over time. Of the focal adhesions disassembled within the period of the movie, 5-6 randomly selected adhesions in 4-5 individual cells were analyzed. Rate constant measurements for disassembly (decreasing fluorescence intensity) of individual adhesions were determined from the slopes of trend lines fitted to semilogarithmic plots of fluorescence intensity ratios over time as described previously38.

6. Supplementary Figure 6 Supplementary Figure 6. A) Western blot analysis showing expression of HPIP in MCF7 and MDA-MB231 cells. GAPDH is used as internal control. B) Representative images of time lapse video microscopy analysis showing the time elapsed between the appearance and dissolution of an observed FAs in MDA-MB231 cells cotransfected with either control sh or HPIP sh1 and DsRed-zyxin plasmids. C) Quantification of time lapse video microscopy analysis of Suppl. Figure 2B. We analyzed 4-5 cells for time-lapse FA disassembly analysis as described previously (Ref. 38). The results are expressed as means ±standard deviation and differences between groups were analyzed by one-way ANOVA.

7. Supplementary Figure 7 Supplementary Figure 7. A) Western blot analysis showing the effect of FAK inhibitor F14 on FAK activation (pFAK Y397) in MCF7 cells. B) Effect of FAK inhibitor F14 (10 µM) on migration of confluent monolayers of MCF7 cells. Representative phase-contrast images of the site at different time points as indicated after scratch wounding were presented. C) Quantification of the kinetics of wound closure assay shown in Suppl. Figure 7B.

8. Supplementary Figure 8 Supplementary Figure 8. A) Effect of calpain2 inhibitor ALLN on migration of confluent monolayers of MDA-MB231 cells. Representative phase-contrast images of the site at different time points as indicated after scratch wounding were presented. B) Quantification of the kinetics of wound closure assay shown in Suppl. Figure 8A. C) Western blot analysis showing the effect of calpain2 inhibitor ALLN (20 µM, 18 hrs) on FAK proteolysis in MDA-MB231 cells. Anti-FAK antibody (Santa Cruz Biotechnology, USA; clone C-20, Cat No: sc-558, antibody specific to C-terminal FAK) could detect unproteolysed (~115 kDa) as well as proteolysed fragments (~35 kDa) of FAK in ALLN untreated sample (lane 1). Although, 35 kDa protein fragment of FAK was also detected in ALLN treated sample (lane 2), the intensity of this protein band was decreased significantly indicating ALLN could inhibit calpain2-mediated FAK proteolysis in MDA-MB231 cells.

9. Supplementary Figure 9 Supplementary Figure 9. In vitro calpain cleavage assay showing the proteolysis of HPIP by calpain2. Bacterial-purified GST-HPIP (5 µg) was incubated in calpain cleavage buffer with the indicated concentrations of purified calpain2 at 30°C for 30 min in presence or absence of CaCl2(1 mM). Western blot analysis detects approx. 45 kDa (GST, 27 kDa plus N-terminal HPIP,18 kDa) protein fragment released from GST-HPIP (FL) (GST, 27 kDa plus full length HPIP, 80 kDa). The asterisk denotes degraded protein products of HPIP during purification/storage.